EP4162039A2 - Nouvelles nucléases crispr omni-59, 61, 67, 76, 79, 80, 81 et 82 - Google Patents

Nouvelles nucléases crispr omni-59, 61, 67, 76, 79, 80, 81 et 82

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Publication number
EP4162039A2
EP4162039A2 EP21818934.8A EP21818934A EP4162039A2 EP 4162039 A2 EP4162039 A2 EP 4162039A2 EP 21818934 A EP21818934 A EP 21818934A EP 4162039 A2 EP4162039 A2 EP 4162039A2
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EP
European Patent Office
Prior art keywords
sequence
seq
amino acid
crispr nuclease
identity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP21818934.8A
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German (de)
English (en)
Other versions
EP4162039A4 (fr
Inventor
Lior IZHAR
Liat ROCKAH
Nadav MARBACH BAR
Nurit MERON
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Emendobio Inc
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Emendobio Inc
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Publication of EP4162039A2 publication Critical patent/EP4162039A2/fr
Publication of EP4162039A4 publication Critical patent/EP4162039A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 5 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 63-90.
  • sgRNA single-guide RNA
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and the CRISPR nuclease comprises a Domain C comprising a sequence having at least 90% identity to the amino acid sequence of at least 90% sequence identity to amino acids 77 to 228 of SEQ ID NO: 5.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and the CRISPR nuclease comprises a Domain D comprising a sequence having at least 90% identity to the amino acid sequence of at least 90% sequence identity to amino acids 229 to 446 of SEQ ID NO: 5.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and the CRISPR nuclease comprises a Domain H comprising a sequence having at least 90% identity to the amino acid sequence of at least 90% sequence identity to amino acids 823 to 921 of SEQ ID NO: 5.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 30-32.
  • crRNA CRISPR RNA
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 6 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 91 and 92.
  • crRNA CRISPR RNA
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7, and wherein the CRISPR nuclease is a nickase formed by an amino acid substitution at D12, E527, H756 or D759 of SEQ ID NO: 7.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 109-120.
  • sgRNA single-guide RNA
  • the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8, and wherein the CRISPR nuclease is a nickase formed by an amino acid substitution at D608, H609 or N632 of SEQ ID NO: 8.
  • the CRISPR nuclease comprises a Domain B having at least 97% sequence identity to amino acids 41 to 76 of SEQ ID NO: 5.
  • the CRISPR nuclease comprises a Domain G having at least 97% sequence identity to amino acids 665 to 822 of SEQ ID NO: 5.
  • the CRISPR nuclease is a nickase formed by an amino acid substitution at D24, E557, H785 or D788 of SEQ ID NO: 1, and effects a DNA break in a DNA strand adjacent to a PAM sequence.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to SEQ ID NO: 3 and the PAM sequence is NRRCM.
  • the CRISPR nuclease is a nickase formed by an amino acid substitution at E584, H585 or N607 of SEQ ID NO: 3, and effects a DNA break in a DNA strand adjacent to a sequence that is complementary to the PAM sequence.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to SEQ ID NO: 5 and the PAM sequence is NGR or NGG.
  • the CRISPR nuclease comprises a sequence having at least 90% identity to SEQ ID NO: 8 and the PAM sequence is NRRNTT.
  • Table 1 lists novel CRISPR nucleases, as well as substitutions at one or more positions within each nuclease which convert the nuclease to a nickase or catalytically dead nuclease.
  • a crRNA molecule of OMNI-61 nuclease may comprise a sequence of SEQ ID NOs: 30-32; a tracrRNA molecule of OMNI-61 nuclease may comprise a sequence of any one of SEQ ID NOs: 33-38; and a sgRNA molecule of OMNI-62 nuclease may comprise a sequence of any one of SEQ ID NOs: 30-39.
  • Other crRNA molecules, tracrRNA molecules, or sgRNA molecules for each OMNI nuclease may be derived from the sequences listed in Table 2 in the same manner.
  • the CRISPR nuclease is a nickase having an inactivated RuvC domain created by an amino acid substitution at a position provided for the CRISPR nuclease in Table 1.
  • the CRISPR nuclease utilizes a protospacer adj acent motif (PAM) sequence provided for the CRISPR nuclease in Table 3.
  • PAM protospacer adj acent motif
  • the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 8 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16 and 24.
  • an engineered or non-naturally occurring composition comprising a CRISPR nuclease comprising a sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease.
  • the CRISPR nuclease is engineered or non-naturally occurring.
  • the CRISPR nuclease may also be recombinant.
  • Such CRISPR nucleases are produced using laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.
  • the composition further comprises a donor template for homology directed repair (HDR).
  • HDR homology directed repair
  • the composition is capable of editing the target region in the genome of a cell.
  • the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 2, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 30-39.
  • the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 5, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 63-90.
  • the CRISPR nuclease and the one or more RNA molecules form a CRISPR complex that is capable of binding to the target DNA sequence to effect cleavage of the target DNA sequence.
  • the nuclease-binding RNA nucleotide sequence is on a first RNA molecule and the DNA-targeting RNA nucleotide sequence is on a single guide RNA molecule, and wherein the first and second RNA sequence interact by base-pairing or are fused together to form one or more RNA molecules or sgRNA that complex with the CRISPR nuclease and serve as the targeting module.
  • the composition further comprises a donor template for homology directed repair (HDR).
  • HDR homology directed repair
  • the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 8, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 16 or 24 and the PAM is NRRNTT.
  • the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 109-120.
  • the CRISPR nuclease is non-naturally occurring.
  • the CRISPR nuclease is engineered and comprises unnatural or synthetic amino acids.
  • the one or more NLSs are in tandem repeats.
  • the composition further comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to the nucleotide acid molecule comprising the sequence encoding the CRISPR nuclease.
  • the CRISPR nuclease or nucleic acid molecule comprising a sequence encoding the CRISPR nuclease is non-naturally occurring or engineered.
  • This invention also provides a non-naturally occurring or engineered composition
  • a vector system comprising the nucleic acid molecule comprising a sequence encoding any of the CRISPR nucleases of the invention.
  • the DNA targeting RNA molecule is a crRNA molecule suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 7, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 15 or 23 and the PAM is NRRAA.
  • the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 99-108.
  • the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell.
  • Domain C begins at an amino acid position within 72-82 and ends at an amino acid position within 223-233 of SEQ ID NO: 5. Based on an analysis of a local alignment generated using the Smith-Waterman algorithm, in an embodiment Domain C has been identified as amino acids 77-228 of SEQ ID NO: 5
  • Domain I is involved in providing PAM site specificity to OMNI-79 nuclease, including aspects of PAM site interrogation and recognition. Domain I also performs topoisomerase activity.
  • a guide RNA molecule designed to target an OMNI- 79 nuclease to a target site is designed to contain a spacer region complementary to a region neighboring a sequence complimentary to the OMNI-79 PAM sequence “NGG.”
  • the guide RNA molecule is further preferably designed to contain a spacer region (i.e. the region of the guide RNA molecule having complementarity to the target allele) of sufficient and preferably optimal length in order to increase specific activity of the nuclease and reduce off-target effects.
  • the guide RNA molecule may be designed to target the nuclease to a specific region of a mutant allele, e.g. near the start codon, such that upon DNA damage caused by the nuclease a non-homologous end joining (NHEJ) pathway is induced and leads to silencing of the mutant allele by introduction of frameshift mutations.
  • NHEJ non-homologous end joining
  • Programmed cell death protein 1 (PD-1) disruption enhances CAR-T cell mediated killing of tumor cells and PD-1 may be a target in other cancer therapies. Accordingly, without limitation, embodiments of the invention that target PD-1 may be used in methods of treating subjects afflicted with cancer. In an embodiment, the treatment is CAR-T cell therapy with T cells that have been modified according to the invention to be PD-1 deficient.
  • BCL11A is a gene that plays a role in the suppression of hemoglobin production. Globin production may be increased to treat diseases such as thalassemia or sickle cell anemia by inhibiting BCL11A. See for example, PCT International Publication No. WO 2017/077394 A2; U.S. Publication No. US2011/0182867A1; Humbert et al. Sci. Transl. Med. (2019); and Canver et al. Nature (2015). Accordingly, without limitation, embodiments of the invention that target an enhancer of BCL11 A may be used in methods of treating subjects afflicted with beta thalassemia or sickle cell anemia.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, in Irons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers,
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • targets refers to preferential hybridization of a targeting sequence or a targeting molecule to a nucleic acid having a targeted nucleotide sequence. It is understood that the term “targets” encompasses variable hybridization efficiencies, such that there is preferential targeting of the nucleic acid having the targeted nucleotide sequence, but unintentional off-target hybridization in addition to on-target hybridization might also occur. It is understood that where an RNA molecule targets a sequence, a complex of the RNA molecule and a CRISPR nuclease molecule targets the sequence for nuclease activity.
  • Eukaryotic cells include, but are not limited to, fimgal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
  • a CRISPR nuclease and a targeting molecule form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence.
  • a CRISPR nuclease may form a CRISPR complex comprising the CRISPR nuclease and RNA molecule without a further, separate tracrRNA molecule.
  • CRISPR nucleases may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule.
  • protein binding sequence or “nuclease binding sequence” refers to a sequence capable of binding with a CRISPR nuclease to form a CRISPR complex.
  • a tracrRNA capable of binding with a CRISPR nuclease to form a CRISPR complex comprises a protein or nuclease binding sequence.
  • Embodiments of the present invention may also form CRISPR complexes utilizing a separate tracrRNA molecule and a separate RNA molecule comprising a guide sequence portion.
  • the tracrRNA molecule may hybridize with the RNA molecule via base pairing and may be advantageous in certain applications of the invention described herein.
  • Any suitable viral vector system may be used to deliver RNA compositions.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids and/or CRISPR nuclease in cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer nucleic acids encoding and/or CRISPR nuclease protein to cells in vitro.
  • nucleic acids and/or CRISPR nuclease are administered for in vivo or ex vivo gene therapy uses.
  • Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Additional exemplary nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc., (see for example U.S. Patent No. 6,008,336).
  • Lipofection is described in e.g., U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787; and U.S. Patent No. 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM., Lipofectin.TM. and Lipofectamine.TM. RNAiMAX).
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, recombinant retroviral, lenti virus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer.
  • an RNA virus is preferred for delivery of the RNA compositions described herein.
  • Nucleic acid of the invention may be delivered by non-integrating lenti virus.
  • RNA delivery with Lenti virus is utilized.
  • the lenti virus includes mRNA of the nuclease, RNA of the guide.
  • the lenti virus includes mRNA of the nuclease, RNA of the guide and a donor template.
  • the lenti virus includes the nuclease protein, guide RNA.
  • the lenti virus includes the nuclease protein, guide RNA and/or a donor template for gene editing via, for example, homology directed repair.
  • compositions described herein may be delivered to a target cell using a non-integrating lenti viral particle method, e.g. a LentiFlash® system.
  • a non-integrating lenti viral particle method e.g. a LentiFlash® system.
  • Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell.
  • a non-integrating lenti viral particle method e.g. a LentiFlash® system.
  • Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • delivery of mRNA in-vivo and ex-vivo, and RNPs delivery may be utilized.
  • nucleic acid template and “donor”, refer to a nucleotide sequence that is inserted or copied into a genome.
  • the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid or may be used to modify the target sequence.
  • a nucleic acid template sequence may be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), preferably between about 100 and 1,000 nucleotides in length (or any integer there between), more preferably between about 200 and 500 nucleotides in length.
  • the donor polynucleotide can be DNA or RNA, single-stranded and/or double- stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 2010/0047805; 2011/0281361; 2011/0207221; and 2019/0330620. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends.
  • a donor template for repair may use a DNA or RNA, single-stranded and/or double-stranded donor template that can be introduced into a cell in linear or circular form.
  • the donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
  • a transgene as described herein may be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to the transgene) or none of the endogenous sequences are expressed, for example as a fusion with the transgene.
  • each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment.
  • any of the RNA molecules or compositions of the present invention may be utilized in any of the methods of the present invention.
  • the sgRNA was predicted by detection of the CRISPR repeat array sequence (crRNA) and a trans-activating crRNA (tracrRNA) in the respective bacterial genome.
  • the native pre-mature crRNA and tracrRNA sequences were connected in-silico with tetra-loop ‘gaaa’ and the secondary structure elements of the duplex were predicted by using an RNA secondary structure prediction tool.
  • At least two versions of possible designed scaffolds for each OMNI were synthesized and connected downstream to a 22nt universal unique spacer sequence (T2, SEQ ID NO: 41) and cloned into a bacterial expressing plasmid under a constitutive promoter and into a mammalian expression plasmid under a U6 promoter (pbSGR2 and pmGuide, respectively, Table 4).
  • the P2A peptide is a self-cleaving peptide which can induce the cleaving of the recombinant protein in a cell such that the OMNI nuclease and the mCherry are separated upon expression.
  • the mCherry serves as indicator for transcription efficiency of the OMNI from expression vector. Expression of all OMNI proteins was confirmed by a western blot assay using anti-HA antibody (Figs. 3A).
  • the editing level obtained by OMNI-79 using the SpSaNX02 scaffold is comparable to the editing level obtained using the native gRNA molecule in all sites tested.
  • the editing level obtained by OMNI-79 using the SpSpCAP1 gRNA molecule is reduced compared with the editing level obtained using the native gRNA molecule.
  • OMNI-79 was subcloned into an AAV packaging construct under a CMV promoter together with a gRNA molecule targeting ELANE g35, CXCR4 or serpinA sl2 under U6 promoter regulation between the ITR components (Table 4).
  • AAV particles were produced by cotransfection of all packaging component plasmids into HEK293 cells, and particles purification (VectorBuilder).
  • AAV particles containing OMNI-79 and a gRNA molecule were used to infect HeLa cells at MOI of 100,000 particles/cell in a 48-well plate format. At 72 hours cells were lysed, and their genomic DNA content was used in a PCR reaction which amplified the corresponding putative genomic targets. Amplicons were subjected to NGS and the resulting sequences were then used calculate the percentage of editing events. As can be seen in Fig 5 A, editing was observed in all sites tested. Editing level using AAV delivery was higher in all three (3) sites, compared with DNA transfection (see table 5).
  • Cell paste was resuspended in lysis buffer (20 mM Hepes, 1000 mM NaCl,50 mM imidazole pH7.5, ImM TCEP) supplemented with EDTA-free complete protease inhibitor cocktail set ⁇ (Calbiochem).
  • Cells were lysed using MC Multi Shot (Constant Systems) French press. Cell disruption was measured by the drop of OD600nm to less than 10% of starting the OD600nm. Clarification of the lysate was done in LYNX6000 centrifuge (Thermo Scientific) at 45,000xg for 30 minutes at 4°C. The cleared lysate was incubated with Ni Sepharose 6 Fast Flow resin (Cytiva).
  • OMNI-79 nuclease Fractions of the OMNI-79 nuclease were pooled and concentrated and loaded onto a centricone (Ami con Ultra ultra 15 5 OK, Merck).
  • the concentrated OMNI-79 protein was further purified by SEC on HiLoad 16/600 Superdex 200 pg-SEC, AKTA Pure (Cytiva) with a 20mM Hepes pH 7.5, 300mM NaCl, 10% glycerol.
  • Fractions containing OMNI-79 protein were pooled and concentrated and loaded onto a centricone (Ami con Ultra 15 5 OK, Merck) with a final storage buffer of 20mM Hepes pH 7.5, 300mM NaCl, 10% glycerol, ImM TCEP.
  • Purified OMNI protein was concentrated to 10-20mg/ml, filtered with SpinX® (Merck), aliquoted, flash-frozen in liquid nitrogen, and stored at -80 °C.
  • RNPs were assembled by mixing 100uM nuclease with 120uM of synthetic guide molecules having different spacer lengths (20-26 nucleotides, Table 6) and lOOuM Cas9 electroporation enhancer (IDT). After 10 mins of incubation at room temperature, the RNP complexes were mixed with 200,000 pre-washed U20S cells and electroporated using Lonza SE Cell Line 4D-NucleofectorTM X Kit with DN100 or program, according to the manufacture’s protocol. At 72 hours cells were lysed, and their genomic DNA content was used in PCR reaction which amplified the corresponding putative genomic targets.

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Abstract

La présente invention concerne une composition d'origine non naturelle comprenant une nucléase CRISPR comprenant une séquence ayant au moins 95 % d'identité avec la séquence d'acides aminés choisie dans le groupe constitué par les SEQ ID NO: 1-8 ou une molécule d'acide nucléique comprenant une séquence codant pour la nucléase CRISPR.
EP21818934.8A 2020-06-04 2021-06-04 Nouvelles nucléases crispr omni-59, 61, 67, 76, 79, 80, 81 et 82 Pending EP4162039A4 (fr)

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AU2015330699B2 (en) 2014-10-10 2021-12-02 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
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JP2023531384A (ja) 2023-07-24
CN116157514A (zh) 2023-05-23
WO2021248016A3 (fr) 2022-04-07
WO2021248016A2 (fr) 2021-12-09
CA3179130A1 (fr) 2021-12-09
KR20230056630A (ko) 2023-04-27
US20250197822A1 (en) 2025-06-19
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